Presence of a particulate thyrotropin-releasing hormone-degrading pyroglutamate aminopeptidase activity in rat liver.

The data of this work describe the presence in the rat liver of a thyrotropin-releasing hormone (TRH)-degrading particulate metal-containing pyroglutamate aminopeptidase of high molecular weight. Following the fractionation of liver homogenate in 0.25 M sucrose, solubilization of the particulate fraction with papain and gel filtration on ACA34, an enzyme activity was detected which converts TRH into pyroglutamic acid and histidyl-proline diketopiperazine (cyclo[His-Pro]; cHP). [L-Histidine-2,5-3H]TRH and [L-proline-2,3-3H]TRH were used as a tracer. Products formed were separated by thin-layer chromatography, localized and quantified by scanning for radioactivity. Enzyme activities were tested using preferential site-directed inhibitors. Particulate pyroglutamate aminopeptidase activity was found to be sensitive to chelating agents. The physicochemical properties of this particulate aminopeptidase were distinct from the soluble pyroglutamate aminopeptidase from the same source. The particulate enzyme shared several similarities with particulate pyroglutamate aminopeptidase from adenohypophysis, brain and with serum enzyme, reported to have narrower specificity for TRH compared to the soluble pyroglutamate aminopeptidase from several tissues. The Km of the gel-filtrated enzyme is 27 microM and the specific activity 306 pmol.min-1.mg protein-1. Although no definite role has been established for this enzyme, it might be a potential determinant of cHP concentrations in liver. Furthermore, cHP is known to possess biological activities and specific binding sites in liver membranes. One of the major sites for TRH breakdown in vivo, the liver, probably represents a target tissue for TRH, especially for pancreatic TRH, and the particulate enzyme involved in the conversion of TRH into cHP may assume a specific function.

Is all cyclo(His-Pro) derived from thyrotropin-releasing hormone?

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is an endogenous cyclic dipeptide that is ubiquitously distributed in tissues and body fluids of both man and animals. This cyclic dipeptide is not only structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), but it can also arise from TRH by the action of the enzyme pyroglutamate amino-peptidase (pGlu-peptidase). The data on the distribution of TRH, cyclo(His-Pro), and pGlu-peptidase under normal and abnormal conditions are summarized and potential relationships analyzed. We conclude that all of the cyclo(His-Pro) cannot be derived from TRH. Two additional sources of cyclo(His-Pro) are suggested. It is proposed that 29,247 molecular weight TRH prohormone, prepro TRH, which contains 5 copies of TRH sequence, can be processed to yield cyclo(His-Pro). Thus, both TRH and cyclo(His-Pro) share a common precursor, prepro[TRH/Cyclo(His-Pro)].

On the mechanism of fasting-associated elevations in hypothalamic cyclo(His-Pro) content.

Potential mechanism(s) underlying the fasting-associated rise in hypothalamic cyclo(His-Pro) content was explored by examining the effects of 24-hour fasting on: (i) cyclo(His-Pro) synthesis from TRH, (ii) cyclo(His-Pro) metabolism, and (iii) cyclo (His-Pro) secretion by hypothalamic tissue in vitro. The data presented here show that none of these three variables were altered due to fasting. Two additional potential changes that could cause cyclo(His-Pro) elevations during fasting are suggested. These include an in vivo decrease in hypothalamic cyclo(His-Pro) secretion that may not be apparent in vitro, and/or an increase in the synthesis of cyclo(His-Pro) from a precursor(s) other than TRH.

In vitro production of a TRH-homologous peptide and His-Pro diketopiperazine by human semen.

Fresh human semen, diluted 1:1 v/v with 0.15 M NaCl- 0.05 M phosphate, pH 7.5, undergoes a 3-fold increase in total thyrotropin-releasing hormone (TRH) immunoreactivity on incubation at 4 C for 8 to 16 hours. To identify the mechanism for this increase, pooled human semen was incubated at 4, 37, and 60 C, and the change in composition of the immunoreactive TRH peptides was quantitated by high pressure liquid chromatography and radioimmunoassay of TRH. His-Pro diketopiperazine, a biologically active metabolite of TRH consisting of a cyclic dipeptide of histidine and proline, also was measured by specific RIA. The concentration of TRH (pGlu-His-Pro-NH2) dropped precipitously within the first hour after dilution and incubation at all temperatures studied. A hydrophobic TRH-homologous peptide with the amino acid composition (Glu,X,Y,Pro), where X and Y are neutral, nonaromatic amino acids, increased 8-fold during 16 hours of incubation at 4 C. This TRH-homologous peptide is not derived from TRH because it lacks the histidine is not derived from TRH because it lacks the histidine residue. A 3- to 23-fold increase in His-Pro diketopiperazine levels occurred after 4 hours at 37 C. This was not due primarily to enzymatic removal of the pyroglutamyl residue from TRH by pyroglutamate aminopeptidase, since about 1 hour after ejaculation the initial His-Pro diketopiperazine levels were 9.7 +/- 5.1 micrograms/ml, or approximately 1000-fold greater than the corresponding levels of seminal TRH. Because cycloheximide, which blocks ribosomal protein biosynthesis, did not inhibit in vitro production of the TRH-homologous peptide and the His-Pro cyclic dipeptide, these peptides, like TRH, most likely arise from post-translational cleavage and processing from pre-existing macromolecular precursor proteins.

Piperazinomycin, a new antifungal antibiotic. I. Fermentation, isolation, characterization and biological properties.

A new antifungal antibiotic, named piperazinomycin, was isolated from the cultured broth of Streptoverticillium olivoreticuli subsp. neoenacticus. The antibiotic was obtained from the mycelial cake by extraction with methanol and also from the broth filtrate by adsorption on Amberlite XAD-2 and subsequent elution with aqueous acetone. The antibitoic is of basic and lipophilic nature and can be extracted with methyl isobutyl ketone at alkaline pH. Its purification was carried out by column chromatography on Sephadex LH-20 and then on Sephadex G-15 followed by preparative thin-layer chromatography on silica gel. The molecular formula of piperazinomycin was determined to be C125H20NsO2 by high resolution mass spectrum and the spectroscopic and chemical properties were examined. Piperazinomycin showed inhibitory activity against fungi and yeasts, especially against Trichophyton.

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis.

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.

The production of chaetoglobosins, sterigmatocystin, O-methylsterigmatocystin, and chaetocin by Chaetomium spp. and related fungi.

Production of mycotoxins by Chaetomium spp. and related fungi on rice culture was examined by a combination of cytotoxicity tests using HeLa cells and thin-layer chromatography. Three species, C. mollipilium, C. rectum, and C. subaffine, as well as C. cochliodes and C. globosum, were proved to produce chaetoglobosins. From cultures of four strains of Chaetomium sp., assigned to C. thielavioideum, and one strain of Farrowia sp., chaetocin, sterigmatocystin, and O-methylsterigmatocystin were isolated. Morphological characteristics of the producers of sterigmatocystins are described.

A30641, a new epidithiodiketopiperazine with antifungal activity.

A new antibiotic, designated A30641, having in vitro activity against Gram-positive bacteria and fungi has been isolated from a strain of Aspergillus tamarii. Chemical and physical characterization indicate that it is a member of the class of antibiotics containing the epidithiodiketopiperazine moiety.

Formation of diketopiperazines by Penicillium italicum isolated from oranges.

Prolyl-2-(1',1'-dimethylallyl)tryptophyldiketopiperazine and 12,13-dehydroprolyl-2-(1',1'-dimethylallyl)tryptophyldiketopiperazine, known metabolites of Aspergillus ustus, were produced in low yield by Penicillium italicum in liquid culture and on unsterilized orange peel.